Engagement of multicomponent immunoreceptors such as the T cell antigen receptor results in rapid recruitment and activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate many enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and then binds many critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, microscopic and genetic techniques are currently employed to study the characteristics of LAT-based signaling complexes and the enzyme pathways that are coupled to and activated at LAT complexes. In the past year we have published studies that focus on different aspects of LAT-mediated signaling. The first can be viewed as a continuation of efforts to bring cutting-edge microscopic techniques to the study of T cell activation. Previously we demonstrated that LAT-based complexes known as microclusters are the site of molecular complex formation occurring with T cell activation. In a study completed this year we used state-of-the-art microscopy including super-resolution analysis to study the molecular organization of these microclusters. We demonstrated that there are two spatially separate domains in the microclusters. One consists of the TCR and associated ZAP-70 PTK and the other contains LAT and LAT-bound signaling molecules. A kinetic analysis of the formation of the microclusters revealed that molecules are recruited in a step-wise fashion beginning with ZAP-70 recruitment to the TCR followed by LAT and other signaling proteins and then by molecules involved in microcluster dissociation. Additionally, this sequence of association is regulated by the calcium elevation that is induced by T cell activation. The sequential steps in the process may be subject to multiple levels of regulation. Over many years we have also studied T cell activation and LAT-mediated signaling by in vitro biophysical analysis of various protein-protein interactions found in signaling complexes. This year we expanded this work to demonstrate the cooperative assembly of a four-molecule complex that normally is formed upon TCR activation. We used recombinant DNA techniques, protein expression and protein synthesis to prepare a doubly phosphorylated LAT peptide, Gads, SLP-76 and phospholipase-Cgamma1. Following mixing of these components, isothermal calorimetry and multi-signal analytical ultracentrifugation were used to determine the composition and thermodynamics of assembly of the heterotetrameric complex. We demonstrated that the four proteins form in a circular arrangement with each protein interacting with two others. Three interactions are of high affinity and one is of low affinity, and this latter interaction may be the site at which formation of this complex is regulated. Our results enable further structural study of this complex.